The overall U19 goal is to develop novel vaccines based on in vivo targeting human dendritic cells (DCs). The focus of the current competitive renewal application is on mucosal immunity. This project will carry out the in vivo immunogenicity and efficacy analysis of human vaccines in humanized mice. Our antibodies are directed against human DC receptors and thus, cannot be tested in regular mice. We have shown the validity of our model for vaccine testing. Thus, systemic and intranasal vaccination with live attenuated virus permits broad influenza virus-specific CD8+ T cell responses. Antigen-specific CD8+ T cells elicited by systemic vaccination can home to the lung. CD8+ T cell immunity can also be generated with recombinant FluM1 protein as well as fusion proteins composed of FluM1 and anti-DC receptor mAbs. Preliminary results indicate the actual targeting of FluM1 antigen to DCs via DCIR, the first demonstration of the targeting of human DCs in vivo. In parallel, we have also analyzed generation of antibody responses and found that vaccination via DCs can lead to generation of influenza-specific neutralizing T-cell dependent IgM that is protective in vivo. While the isotype switch remains a limiting factor in all current models of humanized mice, we are encouraged by our preliminary results which show generation of IgE in mice reconstituted with human stromal cells. Altogether, these results support our current hypothesis: Targeting DCs in vivo with combination of anti-DC mAb-flu antigens fusion proteins and DC activators will generate protective mucosal immunity in humanized mice. AIM 1 will develop our preliminary results and determine the impact of DC activators on skin and mucosal DC activation and antigen internalization. AIM 2 will select vaccines that generate polyfunctional Flu-specific CD8+ T cells with mucosa homing properties. AIM 3 will select vaccines that generate neutralizing mucosal antibodies. AIM 4 will demonstrate in vivo protection of mice vaccinated with polyvalent vaccines selected in vitro (Projects 1 & 2). The final outcome will be protection in challenge experiments upon exposure of vaccinated mice to Flu infection. This project will select fusion proteins and adjuvant combinations that will be tested in the final stage of the project in non-human primates.